Herpes simplex virus offers an important model system for studying gene regulation in human and mammalian cells. Both the early stages of the lytic cycle of the virus and the quiescent latent state of the virus in neurones in vivo must involve complicated and subtle interactions between viral and cellular trans-acting and cis-acting transcriptional factors and control elements. The immediate-early and delayed-early classes of HSV promoters contain many concensus motifs such as the TATA-box, CCAAT-box, octamer, Ap- 1 and Sp-1 sites known to be binding sites for cellular transcription factors. In addition, the four immediate early promoters contain multiple copies of TAATGARAT concensus elements, most of which overlap with octamer-like ATGCTAAT motifs similar to those in immunoglobulin and H2B gene promoters. The TAATGARAT elements are believed to represent specific response sites for an HSV encoded virion transcription factor VF65 (or alpha-TIF), which stimulates the expression of the immediate-early nuclear antigens including the IE175 and IE110 lytic cycle transactivators. The presence or absence of VF65 probably represents a key difference between cells destined for lytic cycle versus those maintaining latent infection. We have shown that both the ATGCTAAT and TAATGARAT motifs bind to a ubiquitous cellular protein NFIII/ORP- C and have tentative evidence that VF65 forms a complex with NFIII when bound to DNA. In this proposal we intend to continue our detailed analysis of the nature of the overlapping octamer/TAATGARAT binding-sites and their interactions with NFIII and VF65 and to precisely identify the signals involved in the trans-activation response. We also plan to map functional domains in the VF65 protein that are involved in trans-activation, and to explore the possibility that cell-cycle specific events may impact on HSV IE expression in various cell types in the absence of VF65.